Effects of serum deprivation, insulin and dexamethasone on polysome percentages in C2C12 myoblasts and differentiating myoblasts

An increase in the rate of protein synthesis in living cells can be achieve d by regulating the quantity of mRNA, ribosomes, and enzymes available for translation or by regulating the efficiency at which existing components ar e used, Efficiency can be measured by comparing the number of ribosomes act ively engaged in the synthesis of protein (polysomes) to the pool of free r ibosomes, The objective of this study was to determine the percentage of ri bosomes found as polysomes in C2C12 cells deprived of serum or exposed to i nsulin or dexamethasone 24 h before and after being stimulated to different iate. Individual 60 mm culture dishes were exposed to serum-free control me dium, medium containing serum, insulin, or dexamethasone for a period of 1 h or 2 h and then quickly frozen. The ribosomes and polysomes from these ce lls were separated by ultracentrifugation on 15 to 60% sucrose gradients an d the absorbance across the gradient at 254 nm was recorded. Polysome perce ntages were determined as the area under the polysome peak divided by the t otal area under the curve, Serum deprivation caused a 12% decline in the pe rcentage of ribosomes found as polysomes (P < 0.01), Dexamethasone caused a quadratic decline (P < 0.05) in polysome percentage, while insulin yielded a quadratic increase (P < 0.05), Protein synthesis assays measuring H-3-ty rosine uptake showed similar responses. These changes occurred in the absen ce of any differences in total RNA concentration. It was concluded that dif ferentiation and the absence of serum in the media reduced the rate of recr uitment of ribosomes for protein synthesis. Insulin increased ribosome recr uitment which was also observed by a similar increase in incorporation of r adio-labeled tyrosine. (C) 1999 Harcourt Publishers Ltd.